1. Field of the Invention
The present invention relates to the field of detection of human papilloma virus (HPV), identifying specific types of HPV, and further to the fields of nucleic acid diagnostics, nucleic acid amplification, and microarrays.
2. Related Art
HPV Types and Classification
To date, over 100 HPV types that infect either cutaneous or squamous epithelia have been identified, and most of these HPV types have been associated with the development of benign or malignant lesions. Through the years different HPV types have been classified according to several criteria such as cutaneous or mucosal types; skin or genital types. However, some discrepancies were seen, as HPV infections identified in some patients did not fit into these specific criteria. Modern classification of HPV is based on the sequence differences that can be identified in the transformation genes E6 and E7 as well as the capsid gene L1 of the virus. A new type of HPV is classified if it shares less than 90% homology for these regions with an already described HPV type. Furthermore, subtypes of HPV consist of types that have 90-98% homology within a genotype, while those with greater than 98% homology within a subtype are described as variants. At present, various types of HPV are categorized according to their association with certain clinical disorders (Table 1). Therefore, HPV types such as HPV-1 and 2 are associated with the development of skin warts, while other types such as HPV-13 and 32 are associated with disorders of the upper respiratory tract.
TABLE 1Classification of HPV according to clinical associationDisorderHPV TypeWarts of the skin1, 2, 3, 4, 7, 10, 26, 27, 28, 29, 41, 48,49, 57, 60, 63, 65Upper respiratory tract2, 6, 11, 13, 16, 32Epidermodysplasia verruciformis5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22,23, 24, 25, 36, 38, 47, 50Anogenital warts2, 6, 11, 16, 18, 30, 40, 41, 42, 44,45, 54, 55, 61Angiogenital carcinomas16, 18, 26, 31, 33, 35, 39, 45, 51, 53,56, 58, 59, 66, 68, 73, 82
Squamous HPV types have been grouped as high-risk or low-risk depending on their transformation capabilities (Table 2) (van den Brule A. et al., 1992), with the high-risk HPV types showing a relative risk (RR) close to 100 for their association with cervical cancer (Duarte-Franco E. & Franco E., 2004). HPV types considered to be high-risk are: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -66 and 69; while HPV-6, -11, -34, -40, -42, -43, and 44 are considered to be low-risk (Kleter, B et al., 1999; Jacobs M V. et al 1995).
TABLE 2Overview of the high-risk and low-risk HPV typesHPV typesHigh-riskHPV-16, -18, -31, -33, -35, -39, -45, -51, -52,-56, -58, -66, -69Low-RiskHPV-6, -11, -34, -40, -42, -43, -44Malignant Transformation by HPV
The two early genes E7 and E6 of high-risk HPV types have been associated with the transformation capabilities of the HPV virus, whereas no such function has been seen for the low-risk HPV types. It has been shown that both genes of high-risk HPV types integrate into the host genome, while those of the low-risk do not (Longworth M and Laimins L., 2004). The integration of the genes results in the disruption of the repressor activity exerted by E2 on the oncoproteins E6 and E7. The mechanisms that are used by these two genes to cause cell transformation have been well investigated and these studies have shown that the early gene E6 inhibits the functions of p53, while E7 interferes with the function of retinoblastoma (Rb) gene.
E6 is thought to degrade p53 at a higher rate than normal through an ubiquitin pathway. E6 recruits a cellular ubiquitin ligase called E6-associated protein (E6AP) with which it forms a complex and replaces the p53 degradation function that is usually carried out by the mdm-2 protein (Longworth M and Laimins L., 2004; zur Hausen H, 2000; Scheffner et al., 1993; Werness et al., 1990). This action is thought to be one of the causes of cancer development as it abolishes the normal function of p53, which is to regulate the expression of proteins that are involved in cell cycle control. The regulation of proteins such as p21, a kinase inhibitor, by p53 results in cell cycle arrest or apoptosis and abolishing these actions can result in carcinogenesis.
Cancer and HPV
The involvement of HPV in the development of cancer mainly cervical cancer has been well documented. This notion was first postulated by Harold zur Hausen and further established with the isolation of HPV 16 from cervical cancer in 1983 (zur Hausen H, 1983). Moreover, HPV is detected in over 90% of cervical cancer. Cervical cancer is one of the most common malignant diseases among women, with more than 500,000 women worldwide being diagnosed with the disease annually (Duarte-Franco E. & Franco E., 2004). Two forms of cervical cancer have been identified, primarily squamous cell carcinoma (SCC) and the rarer cervical adenocarcinoma (AC) (Wang S et. al, 2004). HPV-16 has been predominantly identified in the development of SCC; while HPV-18 has been associated with AC. Precursor lesions, known as cervical intraepithelial neoplasia (CIN) usually precede the development of cervical cancer. These lesions have been classified according to the irregularity in the cells (CIN I), mild to moderate abnormality of the cervix surface lining (CIN II) or full abnormality of the cervix surface lining (CIN III). As with other types of cancer, cervical cancer develops as a result of accumulation of multiple genetic alterations associated to several risk factors. Although risk factors such as smoking, parity, sexual behavior, and the Human Immunodeficiency Virus (HIV) infection have been associated with the development of cervical cancer, the main risk factors are the high-risk HPV (Duarte-Franco E. & Franco E., 2004).
In addition to the involvement of HPV in the development of cervical cancer, it has also been associated with some forms of head and neck squamous cell carcinomas (HNSCC). Over 400,000 cases of HNSCCs are diagnosed annually and studies have shown that in addition to the common risk factors such as smoking and or alcohol consumption, HPV infection might also play a role in this disease. Several studies have been conducted investigating the association of HPV with HNSCCs. One such study performed by Klussmann and colleagues (Klussmann J. et al., 2001) presented evidence of a 26% occurrence of HPV infection in 98 tested HNSCC patients. Furthermore, this study showed that the location of the tumor might also be a factor in the association of HPV infection and cancer development. The frequency of HPV positive cells differs according to the localization of the tumor, with the highest frequency seen in oropharyngeal cancers (45%), and particularly cancer of the tonsil (58%). The HPV type that was frequently detected in these types of cancers was HPV-16; it was detected in 21 of 25 HPV positive tumors. Other HPV types that have been detected are HPV-19 and HPV-33 (Klussmann J. et al., 2001). Other studies performed by McKaig (McKaig et al., 1998) provided evidence of the presence of HPV DNA in 35% of head and neck cancers. These findings were confirmed by Gillison and colleagues (Gillison et al., 2000) with a study performed with 253 tumor samples.
The involvement of HPV in skin cancer was first described in patients with Epidermodysplasia Verruciformis (EV) (Pfister H. 2003; Berkhout R. et al., 2000; Orth G. et al., 1979), who are more susceptible to HPV infection because of a genetic disorder of their immunological system (Weissenborn S. et al., 1999; Berkhout R. et al., 1995). Although several HPV types have been detected in EV patients, HPV types such as 14, 17, 20, 47, 5, and 8 are regarded as high-risk types (Pfister H. 2003; Farve M. et al., 1998; Berkhout R. et al., 1995). Studies have shown that these HPVs are present in high copy numbers and suggest that the infection might be present throughout the development and metastasis of the tumor (Pfister H. 2003).
Current HPV Detection and Typing Techniques
The development of a technique that is capable of detecting and typing HPV efficiently is seen as a possible tool to aid in clinical prognosis and therapy. Most of the current techniques are polymerase chain reactions (PCR) based methods that use consensus primers such as MY09-MY11, PGMY09-PGMY11, and GP5+−-GP6+ to detect HPV, which can be subsequently used in combination with other techniques such as cycle sequencing and dot blots to type or subtype different HPVs (Klaassen C et al., 2004). Although most of these methods have great advantages for being especially sensitive there are also some disadvantages to these methods. The following section will discuss methods that are currently being studied for their use in the detection and typing of HPV.
PCR Based Methods
MY09 and MY11 are degenerate consensus primers that are located in the L1 region of the HPV genome. They consist of 24 pairs of primers that are able to detect more than 30 genital HPV types (Husnjak K et al., 2000; Gravitt P. et al., 2000). This method has been a gold standard in studies to investigate the association between HPV and cervical cancer. However, the results obtained with regard to the sensitivity varied for the different HPV types and the efficiency of the amplification were compromised by the formation of secondary structures (Husnjak K et al., 2000). Moreover, some studies have suggested that the results were frequently irreproducible. As a result of these disadvantages that have been observed an improved set of primers has been generated, PGMY09 and PGMY11, which are a pool of 5 upstream and 13 downstream primers respectively. The primers were designed to bind to HPV types that contain sequence homology in each of the two primer binding regions (Gravitt P. et al., 2000). Studies have shown that the new set of primers improved the sensitivity, specificity, and reproducibility. A similar set of primers to MY09-MY11 was designed by Novelli G. and colleagues (Novelli G. et al., 1992) with the exception of an inosine inclusion at the degenerated positions. They presented the pI-1 and pI-2 primers, which proved in their study to be efficient in the amplification of the L1 region of the cervical cell line CaSki. A study performed by Husnjak K. and colleagues (Husnjak K et al., 2000) showed that the pI-1/2 primers were less sensitive than the MY09-MY11 primers but presented evidence for their possible use in the screening of unknown HPV types. The general primers GP5+ and GP6+ are also designed for the L1 region of the genome and are located within the MY09-MY11 primers (Husnjak K et al., 2000; de Roda Husman A et al., 1995 Snijders P et al., 1990). The primer pair can be used as a two-stepped nested PCR or a one step PCR. In the case of the nested PCR GP5 and GP6 are used in combination with the consensus primer pair MY09/MY11 or PGMY09/PGMY11. A study performed by Evander and colleagues (Evander M et al., 1992) compared both methods and stated that the nested PCR increases the sensitivity of the results. However, the possibility for contamination also increases. These sets of primers are considered more effective than the MY or PGMY primers because the PCR-product generated is of a smaller size, which allows for fewer errors. Another set of primers complementary to the L1 region was designed by Yoshikawa H. and colleagues (Yoshikawa H, et al., 1990). The L1C1, L1C2-1 primers were designed to amplify nine HPV types (HPV-6, -11, -16, -18, -31, -33, -42, -52 and 58) and an additional primer; L1C2-2 amplifying one particular HPV type (HPV-58) was also designed. The different methods that have been mentioned are applied the most, but there are several others such as short PCR fragment (Kleter B. et al., 1998) that are currently used for the detection of HPV. Since the homology between the different HPV types is similar other techniques are combined with these PCR methods to type HPV, including cycle sequencing, line blotting and pyrosequencing (Klaassen C et al., 2004). Table 3 gives an overview of the different primers mentioned.
TABLE 3Overview of different general primersdesigned to the L1 regionPrimerSetSequenceReferenceMY09CGTCCMARRGGAWACTGATCManos et al.,(SEQ ID NO: 1)1989MY11GCMCAGGGWCATAAYAATGG(SEQ ID NO: 2) pI-1GCICAGGGICATAAIAATGGNovelli et al.,pI-2CGTCCIAIIGGAIACTGATC1992(SEQ ID NO: 3) L1C1CGTAAACGTTTTCCCTATTTTTTYoshikawa et al.,(SEQ ID NO: 4)1990L1C2-1TACCCTAAATACTCTGTATTG(SEQ ID NO: 5)L1C2-2TACCCTAAATACCCTATATTG(SEQ ID NO: 6) GP5TTTGTTACTGTGGTAGATACSnijders et al.,(SEQ ID NO: 7)1990GP6GAAAAATAAACTGTAAATCA(SEQ ID NO: 8)
In addition to the techniques mentioned above, there are several other techniques that are being investigated for their usage in HPV detection. One such technique is the Roche AMPLICOR HPV test (Roche Molecular Systems). The test is capable of detecting 13 of the high-risk HPV types and as a positive control the presence of human β-globin is also assessed. The target DNA is amplified and subsequently hybridized for the detection process. A study performed by Monsonego and colleagues (Monsonego J. et al., 2005) concluded that like the HC-II test, the AMPLICOR HPV test, is sensitive enough to detect HPV infection in high-grade lesion. However, they also concluded that the specificity of the test is not as high as the current cytology methods. The AMPLICOR HPV Test amplifies a sequence of nucleotides within the polymorphic L1 region of the HPV genome that is approximately 165 bp in length.
In addition to the AMPLICOR HPV test, a detection/genotyping technique, INNO-LiPa assay was also developed. The INNO-LiPa test (Labo Biomedical Products by, Rijswijk, The Netherlands) is able to detect 25 different HPV types simultaneously and was shown to be sensitive and specific by studies performed by Kleter et al. (1999) and Melchers et al. (1999). This is a short PCR fragment assay (INNO-LiPA HPV detection/genotyping assay, SPF10 system version 1), which amplifies a 65-bp fragment of the L1 open reading frame and allows detection of at least 43 different HPV types.
Hybrid Capture Assay
In addition to the PCR based methods, there are other techniques such as Hybrid Capture (HC) (Digene Corporation, Silver Spring, Md.), which is a hybridization assay that uses RNA probes to type both high-risk and low-risk HPV types.
The Hybrid Capture I (HC-I) was introduced commercially as a non-radioactive assay capable of detecting 14 HPV types (HPV-16, -18, -31, -33, -35, -45, -51, -52, -56, -6, -11, -42, -43 and 44) (Clavel C. et al., 1999; Farthing A. et al., 1995; Schiffman M et al., 1995; Sun X et al., 1995). The technique targets HPV DNA, which are subsequently hybridized with HPV-type specific RNA's. Followed by capture of the DNA/RNA hybrids and signal amplification by binding of the hybrids to multiple conjugated antibodies that specifically recognize DNA/RNA hybrids. The samples are considered positive for high-risk HPV if their assay's chemiluminescence is at least that of the average of three positive assay controls (Snijders et al., 2003, Zielinski G. et al., 2003). A study carried out by Sun XW and colleagues (Sun X et al., 1995) suggested that the test was a sensitive and accurate method to identify high-risk HPV types. However, another study by Clavel C and colleagues (Clavel C. et al., 1999) suggested that the test might not be as sensitive as the current cytology screening technology when it comes to the high-grade lesions. An improved version of HC have been introduced, the HC-II, which is able to detect an additional four HPV types (HPV-39, -58, -59 and 68). Clavel C and colleagues (Clavel C. et al., 1999) performed a study with the HC-II and concluded that the sensitivity of the test was similar to that of PCR using consensus primers and greater compared to cytology screening of high-grade lesions. Although HC shows promising results with an increase in the sensitivity compared to other techniques it also has the disadvantage of providing more false positive results. However, some studies have suggested that because HC is an liquid hybridization test it requires a higher viral load for the detection of HPV DNA (van Ham M. et al., 2005).
DNA Microarray Chips
HPV DNA microarrays have been developed with the notion of being capable of detecting multiple HPV types in a single sample and through one hybridization step. There is currently one commercially available DNA microarray chip, which was introduced by Biomedlab Company, Seoul, Korea. The chip, HPVDNAChip™, contains 22 HPV type specific probes of which 15 are high-risk HPVs (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and 69) and 7 are low-risk HPVs (HPV-6, -11, -34, -40, -42, -43, and 44). For the application of the test, DNA is collected, isolated, subsequently amplified with the general primers GP5 and GP6, and then hybridized to the chip. Studies utilizing the HPVDNAChip performed by groups such as Lee S. et al. (2003) and Jung An H. et al. (2003) gave promising results with high sensitivity detection rate.
Klaassen C and colleagues (Klaassen C et al., 2004) disclose another microarray technique in which they used digoxigenin-labeled HPV-derived PCR amplicons that were hybridized onto biotinylated HPV probes. The hybridized amplicons are then visualized by a staining procedure with a substrate for alkaline phosphatase that has both colomeric and fluorescent properties. Test uses the C terminus of the HPV E1 gene and type-specific probes as well as primers for 53 HPV types. A total of 45 HPV types were identified by a single type-specific probe. It has been proposed that this assay would be more inexpensive than other techniques that are now available. Furthermore, the quantity of HPV types that could be detected with this assay is greater than others and it is also able to detect HPV types that are not yet classified (Klaassen C et al., 2004).
Thus, there are various HPV testing methods available that are being investigated or used to aid in the prognosis and therapy of cervical cancer. These techniques have many advantages but there are also disadvantages. Such is the case for general and consensus primers; the sensitivity percentage is high on one hand but the reproducibility and the ability to detect multiple HPV infection are not optimal (Oh T. et al., 2004). The development of the HPVDNAChip and other DNA microarray has also provided promising results, but the number of types that can be detected are limited such is the case with the HC technique.